Use of R-(Z)!-α-(methoxyimino)-α-(1-azabicyclo 2.2.2.!oct-3-yl)acetonitrile to reduce amyloid βA4 formation in alzheimer&#39;s disease

ABSTRACT

A method of enhancing amyloid precursor protein processing along a non-amyloidogenic pathway in patients suffering from, or at risk of developing, Alzheimer&#39;s disease and a method of treatment or prophylaxis of Alzheimer&#39;s disease by reducing βA4 production in patients suffering from, or at risk of developing, Alzheimer&#39;s disease comprising administering to the patient an effective, non-toxic amount of an acetonitrile compound, use of the compound in preparation of a medicament for use in the method and a composition for use in the method.

This invention relates to a method for enhancing amyloid precursorprotein processing along a non-amyloidogenic pathway and to a compoundfor use in such method.

Amyloid precursor protein (APP) is an integral membrane glycoproteinthat can be processed in several ways. Cleavage by β and γ secretasesultimately leads to release of β amyloid protein (βA4) which isdeposited in the brains of individuals with Alzheimer's disease (AD).Alternative cleavage by α secretase leads to the release ofnon-amyloidogenic APP fragments. The muscarinic agonists carbachol,bethanechol, AF-102B and xanomeline have been shown to enhance theproduction of non-amyloidogenic APP fragments by activation of m1 and/orm3 receptor subtypes (Nitsch et al 1992, Buxbaum et al 1992, 1994,Haring et al 1994, Growdon 1994).

EP-A-0392803 (Beecham Group p.l.c.) discloses certain azabicycliccompounds which enhance acetylcholine function via an action atmuscarinic receptors within the central nervous system, includingR-(Z)!-α-(methoxyimino)-α-(1-azabicyclo 2.2.2!oct-3-yl)acetonitrile(Compound (I)), and pharmaceutically acceptable salts, and processes bywhich such compounds may be made.

WO-93/17018 discloses alternative processes by which Compound (I) may bemade.

It has now been discovered that Compound (I) enhances amyloid precursorprotein processing along a non-amyloidogenic pathway and is therefore ofpotential use in the treatment of Alzheimer's disease by reducing βA4production.

According to the present invention, there is provided the use ofCompound (I) or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for enhancing amyloid precursor proteinprocessing along a non-amyloidogenic pathway in patients suffering from,or at risk of developing, Alzheimer's disease. The invention furtherprovides the use of Compound (I) or a pharmaceutically acceptable saltthereof in the treatment or prophylaxis of Alzheimer's disease byreducing βA4 production.

In a further aspect the invention provides a method of enhancing amyloidprecursor protein processing along a non-amyloidogenic pathway inpatients suffering from, or at risk of developing, Alzheimer's diseasecomprising administering to the patient an effective, non-toxic amountof Compound (I) or a pharmaceutically acceptable salt thereof. Theinvention further provides a method of treatment or prophylaxis ofAlzheimer's disease by reducing βA4 production in patients sufferingfrom, or at risk of developing, Alzheimer's disease comprisingadministering to the patient an effective, non-toxic amount of Compound(I) or a pharmaceutically acceptable salt thereof.

Compound (I) can form acid addition salts with strong acids. The termpharmaceutically acceptable salt encompasses solvates and hydrates.

Compound (I) is preferably provided in a pharmaceutical composition,which comprises Compound (I) or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.

The composition may be in the form of tablets, capsules, powders,granules, lozenges, suppositories, reconstitutable powders, or liquidpreparations such as oral or sterile parenteral solutions orsuspensions.

In order to obtain consistency of administration it is preferred that acomposition is in the form of a unit dose.

Unit dose presentation forms for oral administration may be tablets andcapsules and may contain conventional excipients such as binding agents,for example syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricants, forexample magnesium stearate; disintegrants, for example starch,polyvinylpyrrolidone, sodium starch glycollate or microcrystallinecellulose; or pharmaceutically acceptable wetting agents such as sodiumlauryl sulphate.

Solid oral compositions may be prepared by conventional methods ofblending, filling, tabletting or the like. Repeated blending operationsmay be used to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are of courseconventional in the art. The tablets may be coated according to methodswell known in normal pharmaceutical practice, in particular with anenteric coating.

Oral liquid preparations may be in the form of, for example, emulsions,syrups, or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol, syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminiumstearate gel, or hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles(which may include edible oils), for example almond oil, fractionatedcoconut oil, oily esters such as esters of glycerine, propylene glycol,or ethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid; and if desired conventional flavouringor colouring agents.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, and, depending on theconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the compound can be dissolved in water forinjection and filter sterilized before filling into a suitable vial orampoule and sealing. Advantageously, adjuvants such as a localanaesthetic, a preservative and buffering agents can be dissolved in thevehicle. To enhance the stability, the composition can be frozen afterfilling into the vial and the water removed under vacuum. Parenteralsuspensions are prepared in substantially the same manner, except thatthe compound is suspended in the vehicle instead of being dissolved, andsterilization cannot be accomplished by filtration. The compound can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

The composition may contain from 0.1% to 99% by weight, preferably from10-60% by weight, of the active material, depending on the method ofadministration.

The invention additionally provides a pharmaceutical composition asabove defined for use in enhancing amyloid precursor protein processingalong a non-amyloidogenic pathway in patients suffering from, or at riskof developing, Alzheimer's disease. The invention further provides apharmaceutical composition as above defined for use in the treatmentand/or prophylaxis of Alzheimer's disease by reducing βA4 production.

The dose of the compound will vary in the usual way with the seriousnessof the disorder, the weight of the sufferer, and the relative efficacyof the compound. However, as a general guide suitable unit doses may be5 to 300 μg, for example 10 to 200 μg and such unit doses may beadministered more than once a day, for example two or three times a day,so that the total daily dosage is in the range of about 30 to 600 μg andsuch therapy may well extend for a number of years.

Within the above indicated dosage ranges no unacceptable toxicologicaleffects are indicated for Compound (I).

The following pharmacological data illustrates the invention.

PHARMACOLOGICAL DATA

Amyloid precursor protein processing CHO cells

The effect of test compound on APP processing in Chinese hamster ovary(CHO) cells, transfected with human muscarinic receptors, wasinvestigated using Western blotting techniques. Blots were quantifiedusing a laser densitometer to scan the immunoreactive bands.

Materials

The CHO cells stably transfected with human muscarinic receptors (Bonner1988) were obtained from N.I.M.H. (Md. USA). Tissue culture media andreagents were from Gibco BRL (Scotland). General laboratory reagentswere from Sigma

Chemical Co. (Dorset). R-(Z)!-α-(methoxyimino)-α-(1-azabicyclo2.2.2!oct-3-yl)acetonitrile monohydrochloride is described inEP-A-0392803 as the oxalate salt.

The following primary antibodies were used: 22C11 mouse monoclonalantibody (Boehringer Mannheim, Sussex, UK) which recognises an aminoterminal epitope of APP (Weidemann et al, 1989); anti βA4 1-25 rabbitpolyclonal antibody raised against rat βA4 1-25; Ab54 rabbit polyclonalantibody raised against a carboxy terminal epitope of APP (amino acids751-770 of APP770).

The secondary antibodies were anti rabbit or anti mouse IgG (Sigma Chem.Co., Dorset, UK) followed by rabbit or mouse peroxidase anti peroxidase(PAP) (Sigma Chem. Co., Dorset, UK). The substrate used was the enhancedchemiluminescence kit (ECL) (RPN2106, Amersham, Bucks, UK) andimmunoreactive bands were detected using Hyperfilm-ECL (Amersham, Bucks,UK).

Methods

Cell culture

CHO cells transfected with human muscarinic receptor subtypes hm1, hm2,hm3 and hm4 were grown to confluence on tissue culture dishes (10 cmdiameter) in α-Minimum Essential Medium (αMEM) with ribonucleosides anddeoxyribonucleosides, 10% foetal calf serum, penicillin 100 units/ml andstreptomycin 100 μg/ml.

Cell treatment with test compound

Cells were grown to confluence as above then serum containing medium wasremoved followed by washing the cells twice in 5 ml serum free medium.The cells were then treated with 5 ml serum free medium containing theappropriate concentration of test compound and incubated at 37° C. forthree hours.

The conditioned media was collected, placed on ice and proteaseinhibitors added (1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mMethylenediaminetetraacetic acid (EDTA),1 ug/ml leupeptin). The media wasthen centrifuged for 15 minutes at 3000 G and 4° C. to remove celldebris. The supernatant was removed and concentrated 100 fold usingCentricon 10 concentrators (Amicon, Gloucestershire, UK) by centrifugingat 3000 G for 90 minutes at 4° C. The retentate was collected and storedin aliquots at -20° C. until ready for electrophoresis. The cells werewashed twice with 5 ml serum free medium, scraped and centrifuged for 5minutes at 3000 G and 4° C. to obtain a cell pellet. The supernatant wasremoved and the cell pellet resuspended in 6×volume lysis buffer (0.1MTris pH7.5 containing 1% Triton X100, 5 mM EDTA, 1 mM PMSF, 1 ug/mlleupeptin). The lysate was kept on ice for 30 minutes with intermittentvortexing followed by centrifuging for 5 minutes at 10000 G and 4° C. toremove cell debris. The supernatant was collected and stored in aliquotsat -20° C. until ready for electrophoresis.

Protein Assay

Cell lysate and concentrated culture media samples were assayed forprotein before gel electrophoresis to ensure equal protein loadingbetween treatments on the gel. The proteins were measured by theBradford dye-binding procedure (Bradford, 1976) using Bio-Rad (Herts,UK) dye reagent concentrate. The assay was carried out in a microtitreplate with the absorbance read at 590 nm using a Titretek Multiskan PlusMkII plate reader with software to analyse the results. As variousreagents, particularly detergents, interfere with the Bradford assaycalibrations were carried out in the appropriate sample vehicle.

SDS-PAGE

Proteins were fractionated using a Novex mini-gel system (R & D SystemsEurope Ltd, Oxon, UK). An equal amount of protein was loaded to comparebetween treatments on the same gel. Samples were diluted in 2×reducingsample buffer containing 0.1M Tris pH6.8, 10% sodium dodecyl sulphate(SDS), 0.1% bromophenol blue in glycerol and 50% β-mercaptoethanol. Theproteins were separated on a 6% Tris-glycine polyacrylamide gelcontaining SDS (Laemmli, 1970) for 90 minutes at 100 volts. Proteinstandards were also included of known molecular weight (Sigma).

Western Blot Analysis

After SDS-PAGE the gels were washed for 20 minutes in transfer buffercontaining 2.5 mM Tris, 19.2 mM glycine, 20% methanol at pH 8.3. Theproteins were transferred from the gel onto Immobilon P Polyvinylidinedifluoride (PVDF) membrane (Millipore, Herts, UK) using a Bio-radsemi-dry system for 2 hours at 0.8 mA/cm². After transfer the proteinswere visualised on the membrane by staining with a 10% solution ofPonceau S (Sigma) for 2 minutes. This was destained by rinsing withdistilled water.

The membrane was blocked in phosphate buffered saline (PBS) containing0.1% Tween 20 and 5% dried milk powder for at least 1 hour at roomtemperature. This was followed by two 30 second washes in PBS 0.1% Tween20 (PBST) and then primary antibody was incubated overnight at 4° C. Allantibodies were prepared in PBST containing 2% bovine serum albumin(Sigma). Membranes were washed in PBST for 1 minute followed by two 5minute washes. Secondary antibody, anti-mouse or anti-rabbit IgG(Sigma), was added at 1/3000 or 1/5000 dilution respectively for 1 hourat room temperature. Membranes were washed as above and mouse or rabbitperoxidase anti peroxidase (Sigma) added at 1/3000 dilution for 1 hourat room temperature. A final wash step was in PBS only and thenmembranes were transferred to a clean dish to add ECL substrate(Amersham). The immunoreactive bands were detected on Hyperfilm-ECL(Amersham) which was developed using an automated processor (Kodak).

Densitometric Analysis

Changes in levels of APP were measured using a laser densitometer.Immunoreactive APP bands were scanned using a Pharmacia LKB Ultrascan XLlinked to a computer with Gelscan XL software for processing data. Thelaser beam passed through the sample and the amount of light transmittedwas measured by the photodiode thereby determining how much wasabsorbed. The absorbance readings were integrated to give values of areaunder the curve. Results for vehicle and test compound are expressed asthe mean of 3 experiments±the standard error of the mean (SEM).

Ligand Binding Studies in vitro

Cerebral cortex was dissected from male Hooded Lister rats (Olac, U.K.)into 2.5 volumes (compared with wet weight) ice cold 50 mM tris pH 7.7.This was homogenised then centrifuged at 24,000 g for 15 minutes at 4°C. The pellet was resuspended in 2.5 vols and the homogenates werestored in 1 ml aliquots at -20° C. until required.

Incubations for ³ H!-OXO-M binding were prepared in a total volume of 2ml of ice cold 50 mM Tris, containing 2 mM magnesium chloride. ³H!-OXO-M acetate (New England Nuclear, specific activity 87 Ci/mmol) wasadded to a concentration of 1.88 nM. Cortex homogenate was at a finalconcentration of 300 vols based on the original wet weight (equivalentto 0.145 mg protein/ml). Non-specific binding was defined using 10micromolar (uM) oxotremorine sesquifumarate. Incubations were carriedout to equilibrium at 37° C. for between 30 and 45 mins. Samples werefiltered through Watman GF/B filters pre-soaked for 30 minutes in a0.05% aqueous solution of polyethylenimine to prevent adsorption of ³H!-OXO-M to the glass fibre.

³ H!-QNB, specific activity 44 Ci/mmol, final conc. 0.27 nM) binding wascarried out similarly except that the magnesium chloride was omitted andthe dilution of the homogenate was increased to 1500 vols (7.8 ugprotein/ml). Non-specific binding was defined with 1 uM atropinesulphate.

Binding data for the compound under study are provided in Table 5.

Results

Using the above methods, the effects of the test compound, Compound (I)monohydrochloride, was investigated on amyloid precursor proteinprocessing.

Full length APP with a molecular weight of 108 kD and two bands ofsecreted APP with molecular weights of 108 kD and 118 kD were detectedin all the transfected CHO cells.

CHOhm1

Full length APP in the cell membrane was decreased by Compound (I)monohydrochloride over the dose range 10⁻⁶ -10⁻⁴ M after 3 hourstreatment. In culture medium, test compound increased secreted APP 6-10fold over the concentration range (Table 1).

CHOhm2

There was no change in full length APP in the cell membrane but secretedAPP doubled in culture medium following treatment with 1×10⁻⁴ M Compound(I) monohydrochloride for 3 hours (Table 2).

CHOhm3

Full length APP in the cell membrane was decreased by Compound (I)monohydrochloride over the dose range 10⁻⁶⁻¹⁰ ⁻⁴ M after 3 hourstreatment. In culture medium, test compound increased secreted APP,approximately 20-fold over the concentration range (Table 3).

CHOhm4

There was no change in full length APP in the cell membrane or insecreted APP in culture medium following treatment with 1×10⁻⁴ MCompound (I) monohydrochloride for 3 hours (Table 4).

Ligand Binding Studies

Ligand binding data (Table 5) show that Compound (I) monohydrochloridepossesses a QNB/OXO-M ratio of 22, indicative of partial agonistproperties (Brown et al. 1988).

Conclusion

Compound (I) monohydrochloride alters APP processing by stimulation ofml and m3 receptor subtypes. The enhanced release of secreted APP,particularly as detected with anti βA4 1-25 antibody (which should notrecognise β secretase cleaved APP), strongly suggests this to be along anon-amyloidogenic pathway.

Thus Compound (I) monohydrochloride has potential utility in thetreatment of Alzheimer's disease by reducing βA4 production in the brainas a result of increased non-amyloidogenic APP processing.

REFERENCES

Bonner T (1988) NIH Patent No. PB89-125652; U.S. application Ser. No.7-241 971 filed Aug. 09, 1988

Bradford, M. (1976) Anal. Biochem., 72, 248-254

Brown, F., Clark, M., Graves, D., Hatcher, J., McArthur, R., Riley, G.and Semple, J. (1988) Drug Dev Res, 14, 343-347

Buxbaum, J. D., Oishi, M., Chen, H. I., Pinkas-Kramarski, R., Jaffe, E.A., Gandy, S. E. and Greengard, P. (1992) Proc. Natl. Acad. Sci., 89,10075-10078

Buxbaum, J. D., Ruefli, A. A., Parker, C. A., Cypress, A. M. andGreengard, P. (1994) Proc. Natl. Acad. Sci., 91, 4489-4493

Growdon J. H. (1994) "Muscarinic agonists: Effects on APP processing"4th International Meeting on Alzheimer's Disease, Minneapolis, July1994.

Haring, R., Gurwitz, D., Barg, J., Pinkas-Kramarski, R., Heldman, E.,Pittel, Z., Wengier, A., Meshulam, H., Marciano, D., Karton, Y. andFisher, A. (1994) Biochem. Biophys. Res. Comm. 203(1), 652-658

Laemmli, U. K. (1970) Nature, 227, 680-685

Nitsch, R. M., Slack, B. E., Wurtman, R. J. and Growdon, J. H. (1992)Science, 258, 304-307

Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M.,Masters, C. L. and Beyreuther, K. (1989) Cell, 57, 115-126

In the Tables

Table 1

CHOhm1 Dose Response

Results are the mean of three experiments and expressed as changes fromcontrol APP levels. (A 1-fold increase in secreted APP=no change frombasal). Antibodies used were Ab54 in the cell lysates to detect fulllength APP and 22C11 to detect secreted APP.

Table 2

CHOhm2 Response

The antibodies used were as in Table 1. Results are the mean of threeexperiments and expressed as changes from control APP levels. (A 1-foldincrease in secreted

APP=no change from basal).

Table 3

CHOhm3 Dose Response

Antibodies used were Ab54 in the cell lysates to detect full length APPand anti βA4 1-25 to detect secreted APP. The experiments were carriedout in triplicate. The results are the mean of three experiments andexpressed as changes from control APP levels. (A 1-fold increase insecreted APP no change from basal).

Table 4

CHOhm4 Response

The antibodies used were as in Table 3. Results are the mean of threeexperiments and expressed as changes from control APP levels. (A 1-foldincrease in secreted APP=no change from basal).

                  TABLE 5    ______________________________________    Ligand Binding Data for Compound (I)            .sup.3 H!-OXO-M                        .sup.3 H!-QNB           IC50        IC50     QNB/OXO-M    ______________________________________    Compound (I)*             14            309      22    ______________________________________     *monohydrochloride

OXO-M=Oxotremorine-M, agonist ligand

QNB=Quinuclidinylbenzilate, antagonist ligand

QNB/OXO-M ratio is a guide to the functional efficacy of the compounds.Ratios greater than 100 are associated with full agonists, antagonistsgive ratios close to unity and intermediate values indicate partialagonists.

                                      TABLE 1    __________________________________________________________________________    CHOhm1 Dose Response                       Mean %                 Blot Density                       Decrease in                             Blot Density                                    Mean Fold                 (AUC mm.sup.2)                       Full Length                             (AUC mm.sup.2)                                    Increase in                 (±SEM)                       APP   (±SEM)                                    Secreted APP    __________________________________________________________________________    Control      23.74 ± 6.84                              2.29 ± 1.21    Compound (I)* 1 × 10 - 4M                 14.03 ± 7.27                       40.9  17.54 ± 9.78                                    7.7    1 × 10 - 5M                 12.32 ± 5.91                       48.1  14.48 ± 7.25                                    6.3    1 × 10 - 6M                 16.84 ± 7.20                       29.1   24.2 ± 11.61                                    10.6    __________________________________________________________________________     *monohydrochloride

                                      TABLE 2    __________________________________________________________________________    CHOhm2 Response                Blot Density                      Mean %  Blot Density                                    Mean Fold                (AUC mm.sup.2)                      Decrease in                              (AUC mm.sup.2)                                    Increase in                (± SEM)                      Full Length APP                              (±SEM)                                    Secreted APP    __________________________________________________________________________    Control     12.61 ± 1.0                              2.74 ± 0.55    Compound (I)* 1 × 10.sup.-4 M                12.04 ± 2.56                      4.5     5.75 ± 1.60                                    2.1    __________________________________________________________________________     *monohydrochloride

                                      TABLE 3    __________________________________________________________________________    CHOhm3 Dose Response                 Blot Density                        Mean %  Blot Density                                      Mean Fold                 (AUC mm.sup.2)                        Decrease in                                (AUC mm.sup.2)                                      Increase in                 (± SEM)                        Full Length APP                                (±SEM)                                      Secreted APP    __________________________________________________________________________    Control      23.06 ± 3.78                                 0.85 ± 0.18    Compound (I)* 1 × 10 - 4M                 4.95 ± 1.14                        78.5    15.81 ± 7.87                                      18.6    1 × 10 - 5M                 5.51 ± 1.18                        76.1    15.60 ± 2.27                                      18.4    1 × 10 - 6M                 7.46 ± 3.36                        67.7    16.33 ± 5.37                                      19.2    __________________________________________________________________________     *monohydrochloride

                                      TABLE 4    __________________________________________________________________________    CHOhm4 Response                Blot Density                      Mean %  Blot Density                                     Mean Fold                (AUC mm.sup.2)                      Decrease in                              (AUC mm.sup.2)                                     Increase in                (± SEM)                      Full Length APP                              (±SEM)                                     Secreted APP    __________________________________________________________________________    Control     25.24 ± 0.68                              20.80 ± 4.42    Compound (I)* 1 × 10.sup.-4 M                24.62 ± 0.94                      2.5     22.59 ± 10.28                                     1.1    __________________________________________________________________________     *monohydrochloride

We claim:
 1. A method of enhancing amyloid precursor protein processingalong a non-amyloidogenic pathway in patients suffering from, or at riskof developing, Alzheimer's disease comprising administering to thepatient an effective, non-toxic amount ofR-(Z)!-α-(methoxyimino)-α-(1-azabicyclo 2.2.2!oct-3-yl)acetonitrile or apharmaceutically acceptable salt thereof.
 2. A method of treatment orprophylaxis of Alzheimer's disease by reducing βA4 production inpatients suffering from, or at risk of developing, Alzheimer's diseasecomprising administering to the patient an effective, non-toxic amountof R-(Z)!-α-(methoxyimino)-α-(1-azabicyclo 2.2.2!oct-3-yl)acetonitrileor a pharmaceutically acceptable salt thereof.
 3. A method according toclaim 3 wherein the pharmaceutically acceptable salt is themonohydrochloride.
 4. A method according to claim 2 wherein thepharmaceutically acceptable salt is the monohydrochloride.